We propose to continue to use steroidogenic tissue to determine the regulation and physiologic function of extrahepatic apolipoprotein E. In the past grant period, we have utilized rat and human ovarian granulosa cells to study regulation of apo E because they: 1) utilize large amounts of cholesterol (a known regulator of apo E), 2) respond to multiple regulatory agents, many of which alter cholesterol flow, and 3) are cultured in serum-free medium so that available cell cholesterol can be limited, or provided as lipoprotein. Control of apo E synthesis and secretion by both cholesterol and cell kinase has been demonstrated during this grant period. We will continue these studies, emphasizing the physiological role of extrahepatic apo E, in experiments described in three Specific Aims. In Specific Aim 1, we will utilize immortalized rat ovarian granulosa cells to define kinase, cholesterol, and lipoprotein regulation of apo E secretion. In particular, we will test the ability of cholesterol oxysterol metabolites to act as regulators, and will examine post- transcriptional effects of lipoproteins. In Specific Aim 2, we will determine which cells in the rat tests and ovary produce apo E (by in situ hybridization) and where the apo E localizes after secretion (by immunohistochemistry). We will look for specific apo E movement because of our postulate that gonadal apo E serves both paracrine regulatory and lipid transport functions. The rat testis is an exciting model because one-fifth of the interstitial cells are tissue macrophages. Macrophages are a known source of apo E. In Specific Aim 3, we will continue the study of the physiological role of apo E in the gonad. Using conditioned media and co- culture of different cell groups, we will learn if apo E has paracrine regulatory properties. The gonad is an ideal model because each cell group has a unique steroid product and unique gonadotropin receptors. We can tell which cell is affecting which, not always an easy task in other systems. Lastly, we will use transgenic mice that over-express human apo E to learn the effects of apo E on ovarian follicular development, ovulation, oocyte fertilization and embryo development, and fetal development. These experiments will define the role of local apo E in gonadal tissue, serving the goals of the entire SCOR by serving as a model for the action of apo E at extrahepatic sites.